High density lipoproteins (HDL) are small spherical lipoprotein particles consisting of a neutral lipid core surrounded by a coat of apoproteins, phospholipids and cholesterol. HDL plays a central role in atherosclerotic and coronary diseases. Apoprotein A-I (apo A-I) is the main protein component of HDL. It is thought to be actively engaged in the transport of cholesterol from peripheral tissues to the liver. Our in vivo studies with chickens characterized nascent intracellular HDL particles, showed that lipid-protein assembly occurs mostly in the Golgi apparatus and that proteolytic processing of pro-apo A-I (which contains R.H.F.W.Q.Q at its NH2-terminus) also commences in the Golgi and is completed just prior to, or immediately after, secretion. Studies with cultured chicken hepatocytes and a human hepatocellular carcinoma (Hep G2 cells) showed that intracellular processing occurs in both tissues and that the extent of processing can be regulated by the addition of hormones (and other agents) to the culture medium. Our objectives now are to complete the characterization of the intracellular nascent HDL particles in order to elucidate how the lipid and protein components of HDL are assembled and to understand how the production and secretion of HDL is regulated. The following studies are proposed: 1) The composition of lipid particles presents in the endoplasmic reticulum and the Golgi cell fractions, will be determined by chemical, immunological and immunocytochemical methods. 2) Chicken primary hepatocytes which are known to mimic the physiological state of the donor bird, will be prepared from 5-10 day old chickens which will be subjected to fasting, feeding with cholesterol-rich diet, or to treatment with hormones known to affect lipoprotein metabolism. Apo A-I synthesis, intracellular transport, proteolytic processing, and HDL particle formation will be measured in control and 'prime' hepatocytes. 3) The direct effect of hormones on hepatic HDL synthesis and secretion will be studied using chick embryonic hepatocytes which can be maintained in a chemically defined medium free of added macromolecules.